The Transcriptome of Leishmania major Developmental Stages in Their Natural Sand Fly Vector

ABSTRACT The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi. The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress. IMPORTANCE The life cycle of Leishmania parasites in the sand fly vector includes their growth and development as morphologically distinct forms of extracellular promastigotes found within the different microenvironments of the gut. Based on RNA-Seq, we provide here the first high-resolution, transcriptomic analysis of Leishmania insect stages during their cyclical development in vivo, from tissue amastigotes ingested with the blood meal to infective, metacyclic promastigotes that initiate infection in the mammalian host. The most extensive genetic reprogramming occurred during the early transformation of amastigotes to rapidly dividing procyclic promastigotes in the blood-fed midgut, with major changes in the abundance of mRNAs for surface proteins and metabolism. The post-blood meal-adapted nectomonad stage was characterized by the downregulation of cell cycle-related genes and the upregulation of stress- and starvation-related genes. Finally, the transcriptome of metacyclic promastigotes shifted to a more amastigote-like profile, suggesting their preadaptation to the intracellular host environment. IMPORTANCE The life cycle of Leishmania parasites in the sand fly vector includes their growth and development as morphologically distinct forms of extracellular promastigotes found within the different microenvironments of the gut. Based on RNA-Seq, we provide here the first high-resolution, transcriptomic analysis of Leishmania insect stages during their cyclical development in vivo, from tissue amastigotes ingested with the blood meal to infective, metacyclic promastigotes that initiate infection in the mammalian host. The most extensive genetic reprogramming occurred during the early transformation of amastigotes to rapidly dividing procyclic promastigotes in the blood-fed midgut, with major changes in the abundance of mRNAs for surface proteins and metabolism. The post-blood meal-adapted nectomonad stage was characterized by the downregulation of cell cycle-related genes and the upregulation of stress- and starvation-related genes. Finally, the transcriptome of metacyclic promastigotes shifted to a more amastigote-like profile, suggesting their preadaptation to the intracellular host environment.

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BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

Cancer Cell International ◽

10.1186/s12935-021-01908-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Shuiyan Wu ◽

You Jiang ◽

Yi Hong ◽

Xinran Chu ◽

Zimu Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Acute Lymphoblastic Leukemia ◽

Caspase 3 ◽

Lymphoblastic Leukemia ◽

Rna Seq ◽

Cell Acute Lymphoblastic Leukemia ◽

Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

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Leishmania mexicana Mutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.11.4.1183 ◽

2000 ◽

Vol 11 (4) ◽

pp. 1183-1195 ◽

Cited By ~ 59

Author(s):

James D. Hilley ◽

Jody L. Zawadzki ◽

Malcolm J. McConville ◽

Graham H. Coombs ◽

Jeremy C. Mottram

Keyword(s):

Normal Growth ◽

Surface Proteins ◽

Host Cells ◽

Mammalian Host ◽

Putative Protein ◽

Major Class ◽

Major Surface Proteins ◽

Major Surface

The major surface proteins of the parasitic protozoonLeishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (ΔGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein–anchor precursors. However, the synthesis and cellular levels of other non–protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the ΔGPI8 mutant. Significantly, the ΔGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.

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Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections

10.1101/476994 ◽

2018 ◽

Author(s):

Tom Beneke ◽

François Demay ◽

Edward Hookway ◽

Nicole Ashman ◽

Heather Jeffery ◽

...

Keyword(s):

Life Cycle ◽

Large Scale ◽

Gene Knockout ◽

Sand Fly ◽

Relative Fitness ◽

Lutzomyia Longipalpis ◽

Sand Flies ◽

Label Free ◽

Flagellar Proteins

AbstractThe protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single mutant lacking the central pair protein PF16 showed that these paralysed promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.Author SummaryLeishmania are protozoan parasites, transmitted between mammals by the bite of phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is motile and used for anchoring the parasites to prevent clearance with the digested blood meal remnants. To dissect flagellar functions and their importance in life cycle progression, we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant swimming speeds showed that about half behaved differently compared to the wild type: a few swam faster, many slower and some were completely paralysed. We also found a group of uncoordinated swimmers. To test whether flagellar motility is required for parasite migration from the fly midgut to the foregut from where they reach the next host, we infected sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking these tags up to nine days after infection showed that paralysed and uncoordinated Leishmania were rapidly lost from flies. These data indicate that directional swimming is important for successful colonisation of sand flies.

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H2A.Z is dispensable for both basal and activated transcription in post-mitotic mouse muscles

10.1101/823526 ◽

2019 ◽

Author(s):

Edwige Belotti ◽

Nicolas Lacoste ◽

Thomas Simonet ◽

Christophe Papin ◽

Kiran Padmanabhan ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Start Site ◽

Transcriptional Activity ◽

Muscle Cells ◽

Histone Variant ◽

Start Site ◽

Rna Seq ◽

Transcription Start ◽

Dividing Cells

ABSTRACTThe histone variant H2A.Z is enriched in nucleosomes surrounding the transcription start site of active promoters, suggesting that it might be implicated in transcription. It is also required during mitosis. However, evidences obtained so far mainly rely on correlative evidences obtained in actively dividing cells. We have defined a paradigm in which cell cycle cannot interfere with H2A.Z transcriptional studies by developing an in vivo systems to invalidate H2A.Z in terminally differentiated post-mitotic muscle cells to dissociate its role during transcription from its role during mitosis. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is rather a marker than an actor of transcriptional activity.

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Involvement of Leishmania Phosphatases in Parasite Biology and Pathogeny

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.633146 ◽

2021 ◽

Vol 11 ◽

Author(s):

Anita Leocadio Freitas-Mesquita ◽

André Luiz Araújo Dos-Santos ◽

José Roberto Meyer-Fernandes

Keyword(s):

Neglected Tropical Diseases ◽

Tyrosine Phosphatase ◽

Specific Protein ◽

Sand Fly ◽

Mammalian Host ◽

Metacyclic Promastigotes ◽

Promastigote Form ◽

Parasite Biology ◽

Whole Genome Expression ◽

Drug Resistant Strains

In the Leishmania lifecycle, the motile promastigote form is transmitted from the sand fly vector to a mammalian host during a blood meal. Inside vertebrate host macrophages, the parasites can differentiate into the amastigote form and multiply, causing leishmaniasis, one of the most significant neglected tropical diseases. Leishmania parasites face different conditions throughout their development inside sand flies. Once in the mammalian host, the parasites have to overcome the microbicide repertoire of the cells of the immune system to successfully establish the infection. In this context, the expression of protein phosphatases is of particular interest. Several members of the serine/threonine-specific protein phosphatase (STP), protein tyrosine phosphatase (PTP), and histidine acid phosphatase (HAcP) families have been described in different Leishmania species. Although their physiological roles have not been fully elucidated, many studies suggest they have an involvement with parasite biology and pathogeny. Phosphatases play a role in adaptation to nutrient starvation during parasite passage through the sand fly midgut. They are also important to parasite virulence, mainly due to the modulation of host cytokine production and impairment of the microbiocidal potential of macrophages. Furthermore, recent whole-genome expression analyses have shown that different phosphatases are upregulated in metacyclic promastigotes, the infective form of the mammalian host. Leishmania phosphatases are also upregulated in drug-resistant strains, probably due to the increase in drug efflux related to the activation of ABC transporters. Throughout this review, we will describe the physiological roles that have been attributed to Leishmania endogenous phosphatases, including their involvement in the adaptation, survival, and proliferation of the parasites inside their hosts.

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Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

Infection and Immunity ◽

10.1128/iai.00214-13 ◽

2013 ◽

Vol 81 (7) ◽

pp. 2507-2517 ◽

Cited By ~ 8

Author(s):

Chaoqun Yao ◽

Upasna Gaur Dixit ◽

Jason H. Barker ◽

Lynn M. Teesch ◽

Laurie Love-Homan ◽

...

Keyword(s):

Leishmania Infantum ◽

Parasite Load ◽

Sand Fly ◽

Mammalian Host ◽

Extracellular Medium ◽

Healthy Human ◽

Content Type ◽

Metacyclic Promastigotes ◽

Leishmania Infantum Chagasi

ABSTRACTThe infectious metacyclic promastigotes ofLeishmaniaprotozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. SeveralLeishmaniavirulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). MetacyclicLeishmania infantum chagasipromastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viabilityin vitro. MβCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MβCD-treated promastigotes caused a higher initialin vitroinfection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MβCD-treated promastigotes exposed to healthy human serumin vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence ofLeishmaniaprotozoa at least in part through retention of membrane virulence proteins.

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H2A.Z is dispensable for both basal and activated transcription in post-mitotic mouse muscles

Nucleic Acids Research ◽

10.1093/nar/gkaa157 ◽

2020 ◽

Vol 48 (9) ◽

pp. 4601-4613 ◽

Cited By ~ 5

Author(s):

Edwige Belotti ◽

Nicolas Lacoste ◽

Thomas Simonet ◽

Christophe Papin ◽

Kiran Padmanabhan ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Start Site ◽

Muscle Cells ◽

Histone Variant ◽

Start Site ◽

Rna Seq ◽

Transcription Start ◽

Dividing Cells ◽

Correlational Data

Abstract While the histone variant H2A.Z is known to be required for mitosis, it is also enriched in nucleosomes surrounding the transcription start site of active promoters, implicating H2A.Z in transcription. However, evidence obtained so far mainly rely on correlational data generated in actively dividing cells. We have exploited a paradigm in which transcription is uncoupled from the cell cycle by developing an in vivo system to inactivate H2A.Z in terminally differentiated post-mitotic muscle cells. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is a marker but not an active driver of transcription.

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Multiple Functional Domains of Enterococcus faecalis Aggregation Substance Asc10 Contribute to Endocarditis Virulence

Infection and Immunity ◽

10.1128/iai.01034-08 ◽

2008 ◽

Vol 77 (1) ◽

pp. 539-548 ◽

Cited By ~ 63

Author(s):

Olivia N. Chuang ◽

Patrick M. Schlievert ◽

Carol L. Wells ◽

Dawn A. Manias ◽

Timothy J. Tripp ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Bacterial Load ◽

Rabbit Model ◽

Surface Proteins ◽

Functional Domains ◽

Mammalian Host ◽

Bacterial Cells ◽

Wild Type ◽

Aggregation Substance

ABSTRACT Aggregation substance proteins encoded by sex pheromone plasmids increase the virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis models. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation substance Asc10, encoded by plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the infected tissue was the best indicator of virulence. Isogenic strains carrying either no plasmid, wild-type pCF10, a pCF10 derivative with an in-frame deletion of the prgB gene encoding Asc10, or pCF10 derivatives expressing other alleles of prgB were examined in this model. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative in which glycine residues in two RGD motifs were changed to alanine residues showed the greatest reduction in virulence. Remarkably, this strain and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo.

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A Short-Term Borrelia burgdorferi Infection Model Identifies Tissue Tropisms and Bloodstream Survival Conferred by Adhesion Proteins

Infection and Immunity ◽

10.1128/iai.00349-15 ◽

2015 ◽

Vol 83 (8) ◽

pp. 3184-3194 ◽

Cited By ~ 23

Author(s):

Jennifer A. Caine ◽

Jenifer Coburn

Keyword(s):

Borrelia Burgdorferi ◽

Binding Protein ◽

The United States ◽

Surface Proteins ◽

Mammalian Host ◽

Infection Model ◽

Short Term ◽

Content Type ◽

Extracellular Matrix Components

Borrelia burgdorferi, the causative agent of Lyme disease in the United States, is able to persist in the joint, heart, skin, and central nervous system for the lifetime of its mammalian host.Borreliaspecies achieve dissemination to distal sites in part by entry into and travel within the bloodstream. Much work has been performedin vitrodescribing the roles of manyB. burgdorferiouter surface proteins in adhesion to host cell surface proteins and extracellular matrix components, although the biological relevance of these interactions is only beginning to be exploredin vivo. A need exists in the field for anin vivomodel to define the biological roles ofB. burgdorferiadhesins in tissue-specific vascular interactions. We have developed anin vivomodel of vascular interaction ofB. burgdorferiin which the bacteria are injected intravenously and allowed to circulate for 1 h. This model has shown that the fibronectin binding protein BB0347 has a tropism for joint tissue. We also have shown an importance of the integrin binding protein, P66, in binding to vasculature of the ear and heart. This model also revealed unexpected roles forBorreliaadhesins BBK32 and OspC in bacterial burdens in the bloodstream. The intravenous inoculation model of short-term infection provides new insights into criticalB. burgdorferiinteractions with the host required for initial survival and tissue colonization.

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Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0008671 ◽

2020 ◽

Vol 14 (11) ◽

pp. e0008671

Author(s):

Shaun Wachter ◽

Linda D. Hicks ◽

Rahul Raghavan ◽

Michael F. Minnick

Keyword(s):

Life Cycle ◽

Northern Blot Analysis ◽

Sand Fly ◽

Bartonella Bacilliformis ◽

Phlebotomine Sand Flies ◽

Non Coding Rnas ◽

Srp Rna ◽

Multiple Conditions

Bartonella bacilliformis, the etiological agent of Carrión’s disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión’s disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs αr45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonella bacilliformis small RNA 9 (BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro.

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